1. Field of the Invention
The present invention relates generally to analysis methods wherein it is desired to determine characteristics, such as kinetic properties or affinity for various interactions in multi-component systems. In particular it relates to methods for the analysis of interactions between species in a liquid environment, such as a compound and a target. The invention also relates to the analysis of site specific binding between species, e.g., compounds and targets. More particularly it relates to a method and apparatus for determining kinetic properties or affinity by providing a pulsed gradient of a sample containing a compound of interest, whereby the target molecule is exposed to the gradient of the compound with which it can interact, and detecting a result of said interaction.
2. Description of the Related Art
In the study of candidates for new drugs (screening) it is often the case that substances exhibiting weak binding are encountered, leading to rapid events, exhibiting small time constants. Surface Plasmon Resonance (SPR) is a powerful technique for the study of affinity between substrates and targets, but typically designed for slower events. Instruments utilizing the principle of SPR (e.g., the instruments supplied by the assignee of the present invention, Biacore AB, Uppsala, Sweden) measure changes in refractive index of the medium next to a sensor chip, resulting from altered mass concentration at the surface.
In conventional SPR assays (e.g., using the systems from Biacore AB, Uppsala, Sweden), one sample injection corresponds to one concentration of the selected compound, and the injection comprises one single segment or “plug” of sample liquid. In most cases of kinetic and affinity determination, a few injections of different concentration are sufficient to obtain reliable results of interaction rate or strength (i.e., association rate constant, dissociation rate constant and dissociation constant). However, when studying molecules with low affinity or exhibiting fast kinetics, many such measurements need to be performed. This is a relatively time-consuming process, with considerable sample losses. With the injection exhibiting the highest precision of the available injection methods, every injection requires 40 μl of sample in addition to the desired injection volume to prevent dispersion with buffer.
In an article by Shank-Retzlaff et al, in Analytical Chemistry, Vol. 72, No. 17, pp. 4212-4220, entitled “Analyte Gradient-Surface Plasmon Resonance: A One-Step Method for Determining Kinetic Rates and Macromolecular Binding Affinities”, a method for determining kinetic rates and equilibrium affinities using SPR is disclosed.
It is a one-step method making use of a gradient such that under continuous-flow conditions, the concentration of compound to be analyzed (analyte) passing over the sensor surface increases linearly with time. The rate at which analyte binds to the immobilized receptors is measured by monitoring the change in the surface plasmon resonance minimum as the analyte concentration increases. Kinetic rates are determined by fitting data to a modified version of a two-compartment model.
Although representing an improvement, it still suffers from a lack of capability to perform measurements on systems exhibiting relatively fast kinetic behavior, and also in that relatively large sample quantities are needed for a full titration.